Syndecan binding sites in the laminin alpha1 chain G domain.
Syndecan binding sites in the laminin alpha1 chain G domain.
复制标题
层粘连蛋白 α1 链 G 结构域中的多聚糖结合位点。
作者：
N. Suzuki;Naoki Ichikawa;S. Kasai;M. Yamada;N. Nishi;H. Morioka;H. Yamashita;Y. Kitagawa;A. Utani;M. Hoffman;M. Nomizu
层粘连蛋白α1链G结构域具有多种生物学活性。此前，我们通过筛选113个合成肽-聚苯乙烯小球的细胞黏附活性，确定了层粘连蛋白α1链G区的细胞结合序列。在这里，我们使用了层粘连蛋白α1 G结构域的重组蛋白(rec-α1G)和一大组合成肽来进一步鉴定和表征层粘连蛋白α1链G结构域中的肝素、细胞和Syndecan-4结合部位。Rc-α1G蛋白可促进细胞黏附和肝素结合(K(D)=19 nM)。细胞对rec-alpha1G蛋白的黏附被肝素抑制60%，被EDTA抑制30%。通过竞争肝素与110个合成肽在溶液中与rec-alpha1G蛋白结合来鉴定肝素结合部位。只有AG73(IC(50)=147微米)和AG75(IC(50)=206微米)抑制肝素与rec-α1G的结合。当在固相肝素结合试验中比较这些多肽时，AG73比AG75显示更多的肝素结合。AG73也抑制成纤维细胞与rec-alpha1G蛋白的附着，但AG75不能。用包被多肽的平板和多肽偶联的琼脂糖珠研究了细胞对多肽的附着。AG73在两种实验中均促进细胞黏附，而AG75仅在微珠实验中显示细胞黏附活性。此外，在器官培养中，AG73，而不是AG75，抑制了小鼠颌下腺的分支形态发生。此外，rec-alpha1G蛋白与syndecan-4结合，AG73和AG75均抑制这种结合。这些结果表明，层粘连蛋白α1链G区的AG73和AG75位点对肝素和Syndecan-4的结合是重要的。这些位点可能在涉及肝素和Syndecan-4结合的各种生物学活动中发挥关键作用。
The laminin alpha1 chain G domain has multiple biological activities. Previously, we identified cell binding sequences in the laminin alpha1 chain G domain by screening 113 synthetic peptide-polystyrene beads for cell attachment activity. Here, we have used a recombinant protein of the laminin alpha1 G domain (rec-alpha1G) and a large set of synthetic peptides to further identify and characterize heparin, cell, and syndecan-4 binding sites in the laminin alpha1 chain G domain. The rec-alpha1G protein promoted both cell attachment and heparin binding (K(D) = 19 nM). Cell attachment to the rec-alpha1G protein was inhibited 60% by heparin and 30% by EDTA. The heparin binding sites were identified by competing heparin binding to the rec-alpha1G protein with 110 synthetic peptides in solution. Only two peptides, AG73 (IC(50) = 147 microM) and AG75 (IC(50) = 206 microM), inhibited heparin binding to rec-alpha1G. When the peptides were compared in a solid-phase heparin binding assay, AG73 showed more heparin binding than AG75. AG73 also inhibited fibroblast attachment to the rec-alpha1G protein, but AG75 did not. Cell attachment to the peptides was studied using peptide-coated plates and peptide-conjugated sepharose beads. AG73 promoted cell attachment in both assays, but AG75 only showed cell attachment activity in the bead assay. Additionally, AG73, but not AG75, inhibited branching morphogenesis of mouse submandibular glands in organ culture. Furthermore, the rec-alpha1G protein bound syndecan-4, and both AG73 and AG75 inhibited this binding. These results suggest that the AG73 and AG75 sites are important for heparin and syndecan-4 binding in the laminin alpha1 chain G domain. These sites may play a critical role in the diverse biological activities involving heparin and syndecan-4 binding.